The Role Of HERP On Apoptosis Of Mouse Granulosa Cells

In mammals, the ovary is an extremely dynamic organ that includes follicles in various stages of development. Ovarian follicles develop and progress through the primordial,primary, secondary and antral stages. However, only very few follicles reach the ovulatory stage and subsequently form a corpus luteum, with most undergoing atresia. During folliculogenesis, gonadotropins, such as FSH and LH, play an important role. The pituitary surge of LH/FSH has been thoroughly demonstrated to initiate a complex series of cellular and molecular events in periovulatory follicles leading to the resumption of oocyte meiosis,breakdown of the follicle wall and oocyte release, followed by the subsequent luteinization of the postovulatory follicle. As found in numerous studies, not only endocrine but also paracrine and autocrine factors, which include gonadal steroids, growth factors, cytokines and intracellular proteins, play important roles in ovarian follicular development; for example, an increasing body of evidence indicates that members of the mammalian endoplasmic reticulum(ER) stress response-related molecular chaperones regulate key genes that are crucial for follicular development, atresia and luteinization. To date, the exact molecular mechanisms and interactions between the ER stress response and follicular development remain unclear.The homocysteine-responsive ER-resident protein(HERP) localized in the ER membrane, is induced in the ER stress response. HERP degrades unfolded and misfolded proteins in the ER via the ER-associated protein degradation(ERAD) pathway, and it stabilizes ER Ca2+ homeostasis and maintains mitochondrial function in neuronal cells. HERP is ubiquitously expressed in various organs, such as the heart, liver, skeletal muscle, kidneys and pancreas. Previous studies showed that HERP may be implicated in the pathogenesis of type 2 diabetes, neurodegeneration and sarcopenia. In this study, immunohistochemistry,RNAi, western blot, real time PCR, ELISA, flow cytometry and immunofluorescence were performed to study the distribution, function and regulation mechanism of HERP in the development of follicles. The main results were as follows:(1) The expression and localization of HERP was detected in the mouse ovary ofnewborn, immature and estrous cycle. We found that the expression of HERP was ubiquitous throughout the ovary. High expression of HERP protein was found in the oocytes and granulosa cells; however, the theca cells and corpus luteum exhibited a low HERP expression.The western blot results showed that the expression of HERP was higher at estrous.(2) We designed three RNAi sequences of Herp gene and constructed recombinant shRNA lentiviral vectors successfully. We packaged and determined the titer of recombinant lentivirus using a well-by-dilution titer assay. The final titer of the recombinant shRNA lentivirus was 5-10 × 107 IU/mL. To identify the effect of Herp shRNA, RAW 264.7 cells were transduced with the Herp-shRNA lentiviruses. Herp-shRNA down-regulated the expression of the Herp gene about 80% compared to the control group. We selected the stable expression in knockdown Herp RAW 264.7 macrophages transduced with Herp-shRNA lentivirus. The vectors significantly suppressed the expression of ER stress-related genes treated with the ER stress inducers. However, different ER stress inducers had different effects on inflammatory responses.(3) We detected the roles of Herp in the regulation of the cell cycle, apoptosis and steroid hormone biosynthesis in mouse granulosa cells transduced with Herp-shRNA lentivirus in vitro. After transduction, Herp-shRNA down-regulated the expression of the Herp gene about 70%, the concentration of E2 in the granulosa cell culture medium significantly increased in the Herp-shRNA group compared with the NC-shRNA group. To further confirm the higher release of E2 via Herp knockdown, we analyzed the mRNA expression of steroidogenic enzymes and metabolic enzymes. We found that Herp knockdown significantly increased the mRNA expression of Cyp19a1, which is important for E2 synthesis,and significantly decreased the mRNA expression of Cyp1b1, which is important for E2 metabolism. We also found tnat Herp knockdown arrested cell cycle progression in the Herp-shRNA group compared with the NC-shRNA group by significantly decreasing the m RNA expression of cyclin A1, cyclin B1 and cyclin D2.(4) The apoptotic percentage of granulosa cells was significantly increased by zearalenone(ZEA) treatment in a dose- and time-dependent manner. Moreover, autophagy inhibitor chloroquine(CQ) significantly increased granulosa cells apoptosis treated with ZEA.ZEA induced the expression of the autophagy-related marked protein LC3-II and the ER stress-related proteins GRP78, CHOP and HERP and supressed pERKand pS6 K in MAPK and mTOR signaling pathway in mouse granulosa cells in vitro. ER stress inhibitor 4-PBA significantly supressed the protein levels of ER stress-related proteins and LC3-II in theZEA-induced granulosa cells. CQ significantly increased the expression of LC3-II. We speculated that ZEA induced autophagy by enhancing ER stress and inhibiting MAPK and m TOR signaling pathway.(5) HERP depletion inhibited ZEA-induced cell death in granulosa cells in vitro.ZEA treated increased the expression of LC3-II and BCL-2,and decreased the expression of cleaved Caspase-3 and BAX on the Herp-shRNA group compared with the NC-sh RNA group.We speculated HERP depletion inhibited ZEA-induced cell death in granulosa cells by enhancing autophagy and inhibiting apoptotic signaling pathway.In conclusion, this research studied the distribution, function and regulation mechanism of HERP in the development of follicles. This research provided a new way to study the function of HERP and expanded the research fields of follicles development.